WO1983002896A1 - Procede de purification de facteurs antigenes antiparasitaires - Google Patents

Procede de purification de facteurs antigenes antiparasitaires Download PDF

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Publication number
WO1983002896A1
WO1983002896A1 PCT/US1983/000179 US8300179W WO8302896A1 WO 1983002896 A1 WO1983002896 A1 WO 1983002896A1 US 8300179 W US8300179 W US 8300179W WO 8302896 A1 WO8302896 A1 WO 8302896A1
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Prior art keywords
parasite
antigenic factors
starting
factors
antigenic
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PCT/US1983/000179
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English (en)
Inventor
Antonio Lawrence E D
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D'antonio, Lawrence, E.
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Application filed by D'antonio, Lawrence, E. filed Critical D'antonio, Lawrence, E.
Priority to DE8383901018T priority Critical patent/DE3374270D1/de
Publication of WO1983002896A1 publication Critical patent/WO1983002896A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0233—Rickettsiales, e.g. Anaplasma
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Rodents and primates have been variously vaccinated against malaria with crude plasmodial fragments separated from host blood cells (see e.g., D'Antonio, et al. , Nature,
  • the invention comprises a method for the solubilization and recovery of protective antigenic factors associated with protozoan parasites.
  • the invention further comprises a method for the purification and recovery of protec ⁇ tive antigens of protozoan parasites, particularly parasites of the genuses Plasmodium, Babesia, Trypanosoma, Leish ania, Trichomonas, Entamoeba, Toxoplasma, Pneumocystis, Aegyptianella, Theileria, Anaplasma, and most particularly intraerythrocytic protozoan parasites.
  • the invention additionally provides a vaccine capable of conferring immunity against such parasites comprising the antigenic factors purified and recovered according to the invention.
  • the invention further includes a method for conferring immunity against protozoan parasites comprising administering the parasite antigenic factors purified and recovered according to the invention to a mammal or other vertebrate in immunity-conferring doses.
  • the invention particu ⁇ larly provides a method for the direct extraction of parasite antigenic factors from intact erythrocytes infected .with malarial parasites of the genus Plasmodium, particularly P. berghei, P. malariae, P. viva , P. knowlesi, P. ovale, and P_ ⁇ falciparum, and a method for immunizing mammals or other vertebrates against infection by these parasites.
  • the invention provides a method for diagnosing infection by protozoan parasites.
  • the invention comprises a method for the solubilization and recovery of parasite protective antigenic factors associated with parasite material comprising dispersing the antigenic factors from intact or fractured cells or other tissues infected with protozoan parasites or from free parasite forms with a non-ionic detergent, and separating the solubilized antigenic factors from the dispersing agent and cell or tissue residues.
  • the recovered antigenic factors are useful in vaccines for conferring specific immunity in mammals or other vertebrates to the infecting parasite, or as diagnostic agents.
  • Fig. 1 is a flow sheet illustrating the solubilization and recovery of protective antigenic factors from parasite starting material according to the process of the present invention.
  • Fig. 2 is a flow sheet illustrating the pretreatment of parasite starting material to partially isolate parasite start- ing material components and associated protective antigenic factors.
  • insoluble parasite anti- genic factors associated with insoluble parasite components par ⁇ ticularly the parasite membrane, are recoverable in large quantities from parasite starting material comprising infected intact cells or tissues, partially purified parasite membrane material, or free forms of the parasite.
  • the methods and compositions of the invention are appli ⁇ cable to blood and tissue infecting parasites such as parasites of the genuses Plasmodium, Babesia, Theileria, Aegyptianella, Anaplasma, Trypanosoma, Leishmania, Trichomonas, Entamoeba, Toxoplasma, Pneumocystis, and particularly the Plasmodium species causing malaria such as P. malariae, P.
  • the protective antigenic factors associated with these parasites are recoverable by the process of the present invention from free forms of the parasites in various stages of development, from parasite infected tissues such as liver or skin tissues, or from infected blood, lymph, or other body fluids, particularly red blood cells.
  • plasmodial antigenic factors are recoverable from the sporozoite stage of the parasite by either separating the sporozoites from the host mosquito or isolating them from another environment, or by processing the entire mosquito or culture mixture containing the sporozoites according to the process of the present invention.
  • Plasmodial antigenic factors are also recoverable in purified form from other forms of the parasite such as gametes, microgamates, ookinetes, erozoites, and
  • the starting parasite material useful in the process of the present invention comprises both homogeneous and heterogeneous preparations of different stages or forms of the parasite, either in the absence or presence of unrelated cells or other substances found in typical in vitro cultures or in in vivo host tissues.
  • insoluble protective antigenic factors of these parasites are directly solubilized and recovered from the starting parasite material by solubilizing the protective antigenic factors with a dispersing agent comprising a non-ionic detergent, and separating the solubilized antigenic factors from the insoluble residual material and detergent.
  • the recovered purified antigenic material has enhanced immunoprotective activity owing to both the high concentration of antigenic factors in the recovered material, and, it is believed, " the removal of immuno- suppressive substances produced by the parasite.
  • the parasite material is suspended to the desired concentration in a suitable diluent, such as distilled water or an aqueous isotonic saline solution, and the non-ionic detergent added with agitation to solubilize the insoluble antigenic factors and form a dispersion system having a dispersed phase including the solubilized parasite antigenic factors and the detergent, and an undispersed phase including insoluble parasite material components.
  • a suitable diluent such as distilled water or an aqueous isotonic saline solution
  • the non-ionic detergent added with agitation to solubilize the insoluble antigenic factors and form a dispersion system having a dispersed phase including the solubilized parasite antigenic factors and the detergent, and an undispersed phase including insoluble parasite material components.
  • the dispersed phase is separated from the undispersed phase, conveniently by centrifugation, and the solubilized antigenic factors are separated from the detergent, for example, by ultrafiltration,
  • the reaggregated solubilized antigenic factors are then recovered from the dispersion medium, as by centrifugation.
  • the dispersed phase will also include dispersed foreign material comprising some dispersed parasite components , as well as dispersed tissue or cell components if infected cells or tissues are employed as starting parasite material.
  • the antigenic factors may be further purified, for example in the presence of the dispersing agent by known techniques such as appropriate gel filtration procedures, ion exchange, absorption or affinity chromatography, isoelectric focusing or other electrophoretic procedures, salting out, immunoprecipitation or immunoadsorption using specific antisera or monoclonal antibody preparations, phase separation, or rate zonal or related separation techniques. It may also be desirable to add a protease inhibitor to suspensions of intact cells or tissues prior to rupturing to prevent the possible enzymatic destruction of immunoprotective proteins.
  • compositions employed as diluents for suspending "the starting parasite material and in the filtration procedures are aqueous diluent solutions compatible with the material to be diluted.
  • Distilled water or isotonic salt solutions such as sodium chloride or phosphate buffer are particularly suitable.
  • the pH of the suspension may vary considerably within an exem- plary range of pH 4 -to about pH 8 or 9; it is preferable, however, to maintain the biological material at a pH of about neutral to avoid the possibility of inactivating the desired antigenic factors.
  • Electrolyte salts such as sodium chloride and calcium chloride may be added to the suspending medium so as to result in concen- trations sufficient to bring about optimum solubilization effects in the presence of added detergent.
  • chelating agents such as Ethylenediaminetetraacetic acid (EDTA) may be added to the sus- pending diluent to facilitate detergent solubilization of specific substances.
  • EDTA Ethylenediaminetetraacetic acid
  • the concentration of the parasite starting material in the suspending diluent depends on the nature of the material. Partially isolated parasite starting material is appropriately suspended in concentrations of from about 0.5 to about 5 mg of 5 protein per milliliter, depending upon the state of purification of the starting material; intact cells and tissues are generally suspended in diluent to a concentration of from about 1% to about 50% and most preferably 15% to about 30%.
  • Dispersing agent is added to the suspension of parasite 0 starting material to give a concentration of from about 0.002M to about 0.4M, depending upon the characteristics of the deter ⁇ gent and the suspended material, as well as the concentration of the suspended material.
  • Detergent is added to the starting parasite material preferably in an amount sufficient to obtain 5 optimum activity of the recovered antigenic material; that is, in an amount which maximizes the immunoprotective material solubilized while retaining maximum biologic activity, and which minimizes the foreign material solubilized.
  • deter ⁇ gents of a high extractive efficiency are preferred, especially o such detergents which have little or no tendency to inactivate the protective antigenic factors to be recovered.
  • Suitable dispersing agents are non-ionic detergents such as:
  • Sorbitan monostearate, mono-oleate and -trioleate respectively (SPAN 60, 80 £ 85 respectively).
  • Alkyl phenyl ethoxylate (N0NIDET P40) .
  • a particularly suitable dispersing agent, especially for blood- stage plasmodial antigenic factors is (n-Octy glucos
  • glucoside- detergents such as n-Hexyl- and n-
  • Decylglucoside may also be used alone or in various combinations.
  • the suspension is preferably incubated for a period of time at temperatures which ensure substantial dispersion of the parasite antigenic factors while minimizing bacterial growth and avoiding inactivation of these factors.
  • the dispersing agent is added to the suspension with agitation, and the admixture incubated for from a few minutes to. up to 24 hours or more at temperatures ranging from about 3 C to about 100 C, for example 4 C to 37 C.
  • the dispersed phase is then separated from the undispersed phase, conveniently by centrifugation at 250,000 g maximum at temperatures ranging from about 3 C to about 37 C until the unsolubilized material has aggregated or settled out, usually for about 15 minutes to 1 hour; the number of minutes actually employed will depend primarily upon the density of the suspension. Generally, a pellet of unsolubilized material is obtained, which may be further extracted. The supernatent dispersed phase is then subjected to gel filtration or other separation techniques to remove the detergent and further isolate solubilized antigenic factors.
  • the sol bilized antigenic factors reaggregate are separated from the fraction, as by centrifuging at 250,000 g maximum for about 15 minutes to about 1 hour at from about 3 C to about 10 C.
  • the resultant reaggregation comprises pelletized material, which is then homogenized in a suitable isotonic diluent such as- isotonic saline to a final protein concentration of from about 15 y g/ml to about 200 yg/ml, prefer ⁇ ably from about 33 ⁇ g/ml to about 170 yg/ml, for use as a vaccine.
  • the protective antigenic factors as proteins, there is a direct correlation between pro ⁇ tein content and immunoprotective activity of the recovered material as determined by in vivo assays. If desired, the solubilized antigenic factors may be further purified prior to or after separation of the dispersing agent by known techniques described supr .
  • the pretreatment of intact cells and tissues to partially isolate insoluble plasmodial material prior to dispersion is contemplated.
  • the preteatment of intact cells and tissues broadly comprises the selective disintegration of host cells or tissues while largely preserving the mechanically less fragile parasites, for example, by the use of French Pressure Cells, nitrogen cavitation methods as exemplified by the Parr bomb, the Riby or Hughes press, or ultrasonic techniques.
  • the disintegrated tissue or cellular material is separated from the resulting free parasites or fragments in a series of fractionation and separation steps, and the fraction containing the parasite antigenic factors recovered, usually as isolated partially purified insoluble parasite components, typically membrane material in association with the parasite antigenic factors.
  • This resul ⁇ tant isolated insoluble parasite material in association with parasite antigenic factors is employed as pretreated parasite starting material in the solubilization and recovery process of the invention.
  • low pressure refers to FPC pressures between 800 and 2500 p.s.i. and high pressure to those between 3000 and 40,000 p.s.i.
  • Low gravity refers to centrifugation forces between 50 and 1100 g maximum and high gravity to those between 7000 and 12,000 g maximum.
  • VHG very high gravity refers to centrifugation forces of 200,000 g maximum or less to 250,000 g maximum more or less.
  • the recovered parasite antigenic factors are useful in conferring immunity to infection by specific parasites in mammals and other vertebrates.
  • the dosage ranges will vary, generally depending upon the animal to be immunized, the immunoprotective activity of the recovered antigenic factor fraction, and the specific parasite involved.
  • the immunoprotective antigenic factors have not been purified or identified and reliable in vitro tests for the presence of the antigens are not known to be available, it is very preferably, particularly in the case of vaccine to be administered to humans, that in vivo bioassays such as dose- response studies to be performed on the actual material recovered according to the solubilization and recovery procedure of the present invention to determine the efficacy and toxicity of the recovered material.
  • in vivo bioassays such as dose- response studies to be performed on the actual material recovered according to the solubilization and recovery procedure of the present invention to determine the efficacy and toxicity of the recovered material.
  • dose-response studies are well known, and comprise an evaluation of the immunoprotective activity of the recovered material in an appropriate animal model system, typically rat, mouse or monkey model systems.
  • Sucrose density gradient centrifu ⁇ gation fractions x m A Sucrose density gradient centrifu ⁇ gation fractions x m A.
  • Light density pellet fraction 15 100 100 (PLD)
  • a non-immunizing dose of the antigenic material may be a too high dosage level as well as a too low dosage level.
  • the vaccine is administered by an usual route, parenterally or orally; the vaccine is conveniently administered i.p. in experimental animals, whereas i.v., subcutaneous, or intramuscular routes are preferred for reasons of safety in humans and non- experimental animals.
  • Conventional carriers are employed with the recovered antigenic material for use as a vaccine, such as bio- compatible isotonic salt solutions.
  • the vaccine material may be stored for future use by freezing or lyophilization.
  • the unsolubilized material remaining from the starting preparation following the initial solubilizing step may be reextracted by reapplication of the described procedure or appropriate variations thereof.
  • Isolated antigenic material may also be employed for use in various in vivo and in vitro diagnostic tests. Such tests are useful in the detection, evaluation and following of infections with the related microorganism and for determining immunosensitivity to the involved antigen. The procedures for such tests are well known to those practiced in the discipline.
  • the skin test is an example of an in vivo test. In this test antigen is administered by rubbing into scarified skin; intra- de ⁇ ninal injection; or by application of a patch of material containing the antigen. The skin is then observed at the appropriate time afterwards for reaction indicative of sensitivity to the antigen.
  • an in vitro test is the direct slide agglutination test in which the serum to be tested is added to a suspension or emulsion of aggregated antigen on a glass slide and observed for a clumping of material. Similar type agglutin ⁇ ation tests may be carried out using particulate objects such as erythrocytes, latex or polystyrene spherules or bentonite to which the antigens are first attached or adsorbed. Precipitation in - 13 -
  • fluid media or in gels are examples of other type tests.
  • antisera is added to a solution or gel containing the antigen in soluble form and then observing for a precipitation reaction.
  • fluid precipitation and gel precipitation tests are the interfacial ring test and the Ouchterlony immunodiffusion test respectively.
  • Still other tests using the antigen in appropriate form i.e.
  • soluble or aggregated are the complement fixation test and various antigen or antibody binding tests such as radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and test for cellular sensi ⁇ tivity is the lymphocyte transformation test in which antigenic factors are added to a culture of lymphocytes and the culture than assessed at the appropriate times for the level of * induced lymphocyte transformation.
  • RIA radioimmunoassay
  • ELISA enzyme linked immunosorbent assay
  • test for cellular sensi ⁇ tivity is the lymphocyte transformation test in which antigenic factors are added to a culture of lymphocytes and the culture than assessed at the appropriate times for the level of * induced lymphocyte transformation.
  • the antigenic factors in solution with detergent present may be used in test procedures requiring the antigenic factors in soluble form.
  • Blood infected with the desired specie of malaria is collected in an anticoagulant solution such as heparin or Alsever's solution.
  • the red cells are separated from the plasma by centrifugation in the cold (4 C). at 3500 g for at least five minutes.
  • the plasma and buffy coat is removed by aspiration and the cells are resuspended in a diluent comprising isotonic (0.15M) sodium chloride and recentrifuged. Three additional - washings are employed with the aspiration of any remaining buffy coat each time to assure removal of the white blood cells.
  • the washed cells are resuspended in a volume of
  • NaCl diluent sufficient to give a 20% suspension and then placed in a cooled (4 C) French Pressure Cell.
  • the valve of the French Pressure Cell is at a pressure of about 800 p.s.i.
  • the pres ⁇ sure used for this step depnds on the predetermined optimum pressures for the particular specie of Plasmodium, host red cell, and stage of the parasite; the pressure advantageously is from about 800 p.s.i. to about 2500 p.s.i.
  • the first 2-3 ml of effluent are discarded to avoid contamination with the few unruptured erythrocytes which may initially pass through the needle valve.
  • the above procedure selectively disintegrates the host red cells while largely preserving the mechanically less fragile malaria parasite. While nearly all of the red cells, both infected and non-infected, present in the malarious blood are finely disintegrated, a large number of free intact parasites and large parasite fragments remain. Any intact red cells escaping disintegration may be separated from the French Pressure Cell effluent by centrifugation at between 50 g and 1100 g maximum for ten minutes. - 15 -
  • the supernatant contains the free parasites, large para ⁇ site fragments and a mixture of disintegrated erythrocytes and whatever parasites are disintegrated.
  • the free parasites and large parasite fragments are separated from the disintegrated materials by centrifugation at from about 7000 g to about 12,000 g maximum for up to 30 minutes at temperatures facilitating separation, for example about 3 C to about 10 C.
  • the parasite sediment resulting from the above centrifuga ⁇ tion contains the free parasites and large parasite fragments and is almost completely free of the original host red cell stroma.
  • the sediment A is washed three times by resuspension and centri ⁇ fugation (7000 g to 12,000 g maximum), and finally resuspended in a volume of diluent 8 times the volume of parasite sediment as estimated to give the final desired concentration of parasite vaccine fraction.
  • a volume of diluent 7 to 10 times the volume of parasite sediment will generally result in a final vaccine fraction near that needed for vaccination purposes.
  • the resuspended washed parasite material is passed through the French Pressure Cell at a pressure of 20,000 p.s.i. Pressures from 3000 p.s.i. to 40,000 p.s.i. are suitable. Following the high pressure passage, i.e., 3000 to 40,000 p.s.i., the effluent is centrifuged from about 7000 g to about 12,000 g maximum for 30 minutes at 4 C to remove any disintegrated parasite material E. The resulting supernatant F contains the disintegrated parasite components not sedimenting at the gravity force and time employed. The supernatant F contains the parasite vaccine antigen factors along with a relatively large amount of parasite iron-containing pigment (hemazoin) and other parasite components.
  • hemazoin parasite iron-containing pigment
  • Fraction G is substantially serologically free of host stromal contamination and acts as a specific complement fixing antigen in the serological detection and diagnosis of malaria. Plasmodium berghei and Plasmodium knowlesi derived preparations of G were used to vaccinate mice and monkeys, respectively, against the homologous malaria. Fraction G contains a relatively large amount of hemazoin, which though it does not interfere with the fraction's vaccine or sero- logic properties, must be taken into account when attempting to relate the fraction's protein content to its vaccine concentration.
  • G and PG are immunoprotective and contain membrane material.
  • Product for instance, on ultrastructural analysis (see Reference VI), Product
  • E was found to consist of membranous structures interspersed with what appeared to be membrane bound pigment material (hemazoin) .
  • PG is further fractionated by sucrose density gradient centrfigua ion (see Reference VI) .
  • sucrose density gradient centrfigua ion This is accomplished by resuspension of PG by homogenization into diluent with a teflon-glass homogenizer to a concentration of approximately 0.7 mg of protein per miHiliter , layering on a 20% to 50% performed linear sucrose density gradient, and centrifuging at 217,500 g maximum or two hours at 4 C.
  • Two zones of turbidity develop within the gradient, which upon fractionation, resolves into 260/280 nm absorption peaks in the 20%-22% and 26%-35% sucrose zones respectively. These are respectively designated as the light density (LD) and heavy density (HD) peaks. Dilution and centrif ⁇ ugation of each of these fractions at 250,000 g maximum for 60 - 17 -
  • the LD and HD membranous pellets have been found to protect mice against the homologous malaria infection (see Reference VT) .
  • Preparation LD appears to be signficantly more potent than HD.
  • groups of A/J mice were injected one time intraperitoneally (ip) with 15 ⁇ g of LD or 79 g of HD protein respectively. Eleven and one-half weeks later, the mice
  • Non—treated A/J mice respond to such infection challenge with parasitemias at the end of three weeks ranging from approximately 13% to 69% or higher.
  • the above results affirm the association of the plasmodial protective antigenic factor with the parasite membrane and indicate that preparation LD is a good point of departure for the identification and further purification of such antigenic factors.
  • Bio-Gel A-150 m void volume peak It appears that the soluble protective material is in some way associated with the membrane material and is separated from it by the conditions of prepara ⁇ tion.
  • the procedures of Example I are elaborated in References IV, VI, II, III and U.S. Patent No. 3,849,551, all incorporated herein by reference.
  • mouse red blood cells infected with P. berghei NK65D were used as the source of infective and vaccine material.
  • Isotonic saline was used as diluent throughout and all gel filtrations were carried out at ambient tempertures.
  • Protein determinations were carried out by the method of Lowry (Lowry, et al., J. Biol. Chem. , 193:265-275, 1951) as modified by Yu, et al., Anal. Biochem. , 24:523-530, 1968, except where indicated.
  • Example I describes steps for the pretreatment of parasitic starting material retaining antigenic factors still in association with the insoluble parasite components and, in part ⁇ icular, with the parasite membrane. Although a small quantity of vaccine material appears to separate in soluble form from the parasite during processing, the quantity of material so recovered is small and its potency variable.
  • Example II describes a systematic procedure for effectively separating large quantities of the insoluble parasite antigenic factors in soluble form which permits final purification and characterization of such antigenic factors and offers a practical means for the production of an antimalarial vaccine and related diagnostic agents. Highly active malaria antigenic factors were effectively solubilized from - 19 -
  • IIB isolated insoluble plasmodial material
  • IIC intact plasmodially infected RBC
  • the A/J mouse model malaria vaccination system (see Reference I) was used for the detection and comparative immunogenic evaluation of solubilized recovered plasmodial antigen(s) .
  • CF-1 mice served as a source of infected and non- - infected blood.
  • the reticulocy e-infecting Plasmodium berghei strain NK65 (Line D) obtained from the University of Illinois, Urbana, Illinois, served as a source of plasmodial protective a ⁇ d infective material.
  • the non-ionic dispersing agents employed are generally compatible with biochemical and immunochemical separation procedures.
  • the non-ionic detergent n-Octyl-glucoside used in this exemplary process is an excellent exampel of such dispersing agents.
  • highly active malaria anti ⁇ genic factors were effectively solubilized from isolated insoluble plasmodial material (B) and intact plasmodially infec ⁇ ted red blood cells (C) .
  • Isolated P. berghei NK65D plasmodial material represented by fraction E of Example I was homogenized in iso ⁇ tonic saline diluent with a teflon-glass homogenizer to a concentration of 1.2 mg of protein per.milliliter.
  • the suspension was placed in a beaker and rapidly stirred with a magnetic bar stirrer while n-Octylglucoside powder was slowly added to the final concentration of 0.03M (6.75 mg detergent per mg protein present) .
  • the mixture was incubated at 4°C for four hours and then centrifuged at 250,000 g maximum for 30 minutes at 4 C to remove unsolubilized material.
  • the resulting pellet was set aside for repeated extractions and the clear supernatant eluated - 20 -
  • Washed mouse red blood cells which were 19% infected with malaria parasites were suspended to 20% in isotonic saline containing 0.03 M n-Octylglucoside (44 mgm of glucoside per milliliter of packed red cell equivalent) and incubated overnight at 4 C. Following incubation, the unsolubilized material was - 21 -
  • Respective groups of A/J mice were injected one time ip with one milliliter of preparation PSE or respective concentrations of PSR as shown in Table 1. Eight to eleven weeks later, the treated mice along with non-treated control
  • mice were challenged ip with 10 plasmodially infected mouse red blood cells and parasitemia levels for each mouse determined weekly. Successfully vaccinated mice responded with low level parasitemias which began to resolve by the second and third weeks post challenge. Non-treated mice, on the other hand, experience progressively increasing parasitemias, which did not begin to resolve, in those destined to survive, before the fifth week post challenge.
  • mice receiving PSE Group II-A were still alive at the end of three weeks post challenge and all were without detectable parasitemias.
  • the pattern of survival and parasitemia by the third week post challenge for the groups of mice receiving various concentrations of PSR were dose related.
  • 100% of mice receiving 172 ⁇ g to 208 ⁇ g of preparation PSE protein were still alive.
  • mice in each group with third week parasitemias of less than 1% were 100%, 63% and 9%, respectively, for those receiving the high, intermed ⁇ iate or low dose respectively.
  • 79% of the non-treated control mice were still alive.
  • Parasitemia levels in these animals ranged from 13% to 57% (mean 32% j- SD 12) .
  • a vaccine was prepared by incubation of a 30% suspension of infected mouse red blood cells in a 0.07 M n-Octyl- glucoside solution (70 mg of glucoside per packed cell equivalent) for 30 minutes at 4 C. The remainder of the vaccine recovery pro ⁇ cedure was according to Example II (C) for processing intact infected blood.
  • the rapidly attained vaccination response indicates that the vaccine material isolated as described is unusally potent and that the solubilization and recovery technique produces antiparasitic vaccine preparations with greatly enhanced activit .
  • the solubilization and recovery process of the present invention allows for recovery of antigenic factors which can function as vaccine or diagnositic agents or both.
  • the pro ⁇ cedures described, along with any number of variations, will allow for the practical separation and purification of specific antigens for the first time and opens the way for their use in a variety of immunological, immunobiological and immunodiagnostic ways.
  • Antiparasitic vaccines developed by the procedures described may be individual vaccines for each specie of parasite to be protected against or a combination of various species to form polyvalant vaccines.
  • the vaccines could, in addition, be composed of antigenic factors from different stages of the parasite so as- to form a " ulti" vaccine.
  • blood stage malaria or other parasite vaccines could be made up of a combination of anti ⁇ genic factors derived from different lots of parasites of the same specie in order to insure protection against heterologous strain variants, should they exist.
  • the various antigenic factors once isolated, could also be biochemically altered so as to increase their immunization potency, should this prove necessary. Such alterations could range from intrinsic changes in the molecular structure of such anti- genic factors to coupling them to powerful immunostimulating carrier molecules.
  • the vaccine antigenic factors themselves may eventually be either partially or completely synthesized by bio ⁇ chemical or recombinant DNA techniques. As with the naturally - 25 -
  • derived antigenic factors those produced by such synthetic techniques could also be separated from the "matrix" with which they may be associated by appropriate application of the pro ⁇ cedures described.

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  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Procédé de solubilisation et récupération des facteurs antigènes insolubles associés à des matériaux de parasites et offrant une protection contre ces parasites. Le procédé consiste à solubiliser les facteurs antigènes avec un détergent non-ionique et à séparer le matériau solubilisé du matériau résiduel non dispersé. Les facteurs antigènes protecteurs purifiés sont utilisables comme vaccins, notamment contre la malaria, et comme agents diagnostiques.
PCT/US1983/000179 1982-02-17 1983-02-09 Procede de purification de facteurs antigenes antiparasitaires WO1983002896A1 (fr)

Priority Applications (1)

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DE8383901018T DE3374270D1 (en) 1982-02-17 1983-02-09 Method for the purification of parasite antigenic factors

Applications Claiming Priority (2)

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US34961682A 1982-02-17 1982-02-17
US349,616820217 1982-02-17

Publications (1)

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WO1983002896A1 true WO1983002896A1 (fr) 1983-09-01

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PCT/US1983/000179 WO1983002896A1 (fr) 1982-02-17 1983-02-09 Procede de purification de facteurs antigenes antiparasitaires

Country Status (6)

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EP (1) EP0101505B1 (fr)
AU (1) AU582112B2 (fr)
CA (1) CA1230550A (fr)
DE (1) DE3374270D1 (fr)
NZ (1) NZ203292A (fr)
WO (1) WO1983002896A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1984002917A1 (fr) * 1983-01-28 1984-08-02 Inst Medical W & E Hall EXPRESSION DE POLYPEPTIDES DE PLASMODIUM FALCIPARUM A PARTIR D'ADNc
WO1986006075A1 (fr) * 1985-04-11 1986-10-23 The Walter And Eliza Hall Institute Of Medical Res Antigenes hautement repetitifs de plasmodium falciparum
US4859464A (en) * 1982-02-17 1989-08-22 Antonio Lawrence E Method for the purification of parasite antigenic factors
US5032397A (en) * 1982-12-27 1991-07-16 Institut Pasteur Polypeptidic fractions inducing protective antibodies against malaria parasites and immunogenic compositions
EP0471293A2 (fr) * 1990-08-15 1992-02-19 Abbott Laboratories Réactif de solubilisation pour des spécimens biologiques
US5101017A (en) * 1987-04-06 1992-03-31 New York Blood Center, Inc. Antibodies for providing protection against P. vivax malaria infection
WO1998009637A2 (fr) * 1996-09-02 1998-03-12 Rademacher Group Limited Traitement du diabete

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
NZ232279A (en) * 1989-02-01 1991-11-26 Univ Melbourne Method for producing antibody which involves isolating cells from a sample and culturing; production of antigens using the antibody produced; diagnostic kits and vaccines

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EP0003529A1 (fr) * 1978-01-25 1979-08-22 The Wellcome Foundation Limited Antigènes de Trypanosoma cruzi, leur purification et leur utilisation dans des vaccins
US4298596A (en) * 1979-03-29 1981-11-03 Burroughs Wellcome Co. Trypanosoma cruzi glycoprotein vaccine for inducing immunity to Chagas' disease
FI64813C (fi) * 1980-12-31 1984-01-10 Ilkka Antero Palva Foerfarande foer producering av ett utvalt aeggviteaemne och vid foerfarandet anvaenda rekombinantplasmidvektorer
IL65496A (en) * 1981-04-15 1985-10-31 Wellcome Found Protozoal antigen,its preparation and vaccine containing it
EP0071705B1 (fr) * 1981-05-21 1989-11-23 The Wellcome Foundation Limited Antigène protozoaire

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Title
CHEMICAL ABSTRACT, Volume 88, No. 21 issued 1978 (Columbus Ohio, U.S.A.), SIDDIQUI et al., "In Vitro Cultivation and Partial Purification of Plasmodium Falciparum Antigen Suitable for Vaccination Studies in Aotus Monkeys". Sse page 417, column 1, the Abstract No. 150301e, J. PARASITOL. 1978, 64(1), 168-9 (Eng). *
Infection and Immunity, Volume 28, No. 1., issued 1980 (United States), GROTHAUSand KREIER, "Isolation of a Soluble Component of Plasmodium Berghei Which Induces Immunity in Rats". see pages 245-253 *
Proc. Natl. Acad. Sci., Volume 77, No. 6., issued 1980 (United States), KILEJIAN, "Stage-Specific Protein and Glycoproteins of Plasmodium Faliciparum: Identification of Antigens Unique to Schizonts and Merozoites". see page 3695-3699 *
See also references of EP0101505A4 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4859464A (en) * 1982-02-17 1989-08-22 Antonio Lawrence E Method for the purification of parasite antigenic factors
US5032397A (en) * 1982-12-27 1991-07-16 Institut Pasteur Polypeptidic fractions inducing protective antibodies against malaria parasites and immunogenic compositions
WO1984002917A1 (fr) * 1983-01-28 1984-08-02 Inst Medical W & E Hall EXPRESSION DE POLYPEPTIDES DE PLASMODIUM FALCIPARUM A PARTIR D'ADNc
GB2143830A (en) * 1983-01-28 1985-02-20 Inst Medical W & E Hall Expression of plasmodium falciparum polypeptides from cloned cdna
WO1986006075A1 (fr) * 1985-04-11 1986-10-23 The Walter And Eliza Hall Institute Of Medical Res Antigenes hautement repetitifs de plasmodium falciparum
US5101017A (en) * 1987-04-06 1992-03-31 New York Blood Center, Inc. Antibodies for providing protection against P. vivax malaria infection
EP0471293A2 (fr) * 1990-08-15 1992-02-19 Abbott Laboratories Réactif de solubilisation pour des spécimens biologiques
EP0471293A3 (en) * 1990-08-15 1992-03-18 Abbott Laboratories Solubilization reagent for biological test samples
WO1998009637A2 (fr) * 1996-09-02 1998-03-12 Rademacher Group Limited Traitement du diabete
WO1998009637A3 (fr) * 1996-09-02 1998-05-14 Univ London Traitement du diabete
US6214357B1 (en) 1996-09-02 2001-04-10 Rademacher Group Limited Treatment of diabetes

Also Published As

Publication number Publication date
DE3374270D1 (en) 1987-12-10
CA1230550A (fr) 1987-12-22
AU582112B2 (en) 1989-03-16
AU1376683A (en) 1983-09-08
NZ203292A (en) 1986-07-11
EP0101505A4 (fr) 1984-07-06
EP0101505B1 (fr) 1987-11-04
EP0101505A1 (fr) 1984-02-29

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